JPH034793A - Lysozyme gene from streptomyces, method for obtaining same and its use - Google Patents

Abstract

NEW MATERIAL: A lysozyme gene of Streptomyces origin (a) capable of hybridization with an oligonucleotide probe with 5'-TTC GC (G/C) TAC ATC AAG GC(G/C) AC(G/C) GAG GG(G/C) AC(G/C) AAC TAC AA-3' sequence, and/or (b) after transformed, complementary to lysozyme-deleted mutant Streptomyces coelicolor DSM 4913.

USE: Production of bacterial lysozyme.

PREPARATION: This lysozyme gene is obtained by genetic manipulation from Streptomyces, pref. Streptomyces coelicolor (Mueller) or Streptomyces labedae, or mutant or variant thereof.

COPYRIGHT: (C)1991,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of Streptomyces
etes), the transfer of this gene into various microbial strains and the preparation of gene products. Modification of genes by replacement of surrounding signal sequences has also been described.

Enzymes defined as lysozymes include the C
1 and C4 of N-acetylglucosamine of bacterial peptidoglycan [
Jolles, P., and Jolles, J.:
Molecular and CellularBio
chemistry 63.1 (35-189 (198)
4)]. Lysozyme has been discovered and studied in many organisms. The amino acid sequence is known and the reaction mechanism was proposed for lysozyme from egg albumin (Jo
Les et al.: Biochim, Biophys
, Acta 78.668-6689.1963, P
h1llips, D, C,: Sci, Am,
215.78-90.1966゜ The review is by Jo
See Jolles and Jolles. ), other examples of species in which lysozyme has been detected include Strept.
omyces Iabedae (Streptomyc
es “erythraeus”) [:Morita
et al: J.

13iochem, 83, 893-903, (
1976) Ko, Stereptomycesgri
seus [Ward and Perkins: 8io
chem, J.

106, 6 Fu 76, (1968) Ko, St
reptomycesglobisporus [Ka
wata et al.: Agric, Biot, Chem.

47, t501-1508, (1983),
StreptOmyCeSrutgerssensi
s [Hayashi et al.: J, Ferment.

Technol, 59.319-323 (1981
) and Streptomyces coelico
lor (Mjller) (DE3440735).

With the development of genetic engineering methods, the cloning and detailed analysis of various lysozyme genes has become possible. As an example, the gene for lysozyme from egg albumin (J
uyen-Huu et al.: Proc.

Natl, Acad, Sci, USA 76.7
6-80.1979) and lysozyme from phage T4 (Qwen et al.: J.

Mo1. Biol, 165.229-248.19
83) etc.

Furthermore, DNA chemical synthesis methods have been used for de novo synthesis of the lysozyme gene. References include Novel Synthesis of the Human Lysozyme Gene (EP0181634, Ike
Hara et al.: Chem, Pharm, Bull,
34.2202-2208.1986) and its expression in microorganisms (EP 0255233, Murak
i et al.: Agric, Riot.

Chem, 49.2829-2831.1985, Yo
Shimura et al. Biochem, Bioph
ys, Res, Co1n, 150.794-80
1°1988). In addition, new genetic manipulation methods have been developed to specifically modify the naturally occurring lysozyme gene (Perry, L, J., and Wetze et al.
L, R, et al.: 5science 226, 555-55
7.1984, Wetzel et al.: Proc, Na
tl, Acad, Sci, 85.401-405, 19
88, Muraki et al.: Biochim, Bio
phys, Acta 916.66-75.1987
) and thereby have been used to obtain information about the reaction mechanism and to influence enzyme properties, such as temperature stability, for example.

A gene encoding lysozyme has now been found in Streptomyces. The present invention therefore relates to a lysozyme gene from Streptomyces, which comprises a) the following sequence 5'-TTCGC(G/C)TACATCAAG GC
(G/C) AC (G/C) GAG GG (G/C)
AC(G/C) hybridizes with the oligonucleotide probe of AACTACAA-3′, and/or b) After transformation, the lysozyme-negative mutant Str
eptomyces coelicolor DSM
This is a supplement to 4913.

The lysozyme gene derived from a microorganism according to the present invention is derived from Streptomyces, preferably Streptomycesco
elicolor (Muller) and Star
ptomyces Iabedae, or mutants and variants of these seedlings. DSM 3030 strain (S.

Coel 1color) and DSM 41517 strain (S, 1abedae) are particularly preferred.

Total DNA is isolated from these strains using conventional methods and cut into fragments of various sizes.

This can then be ligated with a cosmid or plasmid vector before transformation of the host strain. Alternatively, the chromosomal DNA fragment of interest can be prepositioned by hybridizing digested total DNA with radiolabeled oligonucleotides.

In this case, the sequence of the oligonucleotide used must correspond to the amino acid sequence in the lysozyme protein determined beforehand according to the rules of the genetic code.

It is then necessary to identify a host clone containing the lysozyme gene from a suitable gene bank. The steps depend on the host strain used and are as described below.

Expression of Streptomyces genes with natural signal sequences is extremely poor in E. coli, making it difficult to use activity assays to identify lysozyme genes cloned into E. coli. is not a good idea.

Therefore, significant expression of lysozyme activity in E. coli cannot be expected, and in fact, it may be toxic to cells. However, by establishing the partial amino acid sequence of lysozyme cleaved into fragments and then using oligonucleotide probes, the desired clone can be found. The specific amino acid sequence then yields related nucleotide sequences at the DNA level, according to the rules of the genetic code. A given amino acid can be determined by up to six different codons.

For established amino acid sequences, a search is performed for regions with sequences of amino acids that can be determined at the DNA level with a minimum number of codons.

The corresponding oligonucleotide sequences are deduced from these amino acid sequence regions. Due to the degeneracy of the genetic code, the number of possible nucleotide sequences increases rapidly. A sequence of about 20 nucleotides is generally sufficient for this purpose.

In preliminary tests, this type of probe gave some fairly specific signals, but
In practice, these probes proved unsuitable for gene detection.

The only one that was used successfully, both in preliminary testing and in the screening steps described below, was the 38 nucleotide DNA TTCGC(G/C)TACATCA.
AG GC (G/C) AC (G/C) GAG GG
(G/C)AC(G/C)AACTACAA-3'. Streptomyces DNA
Due to the high G/C content of more than 70% in the probe synthesis, only G or C was used in the third position of the codon used in the probe synthesis. With this probe,
S the DNA band that specifically hybridizes.

Complete DNA of coelicolor (Mueller)
as well as other Streptomyces strains, e.g.
It can also be identified in the total DNA of S. abedae.

Available plasmid or cosmid gene bank clones are examined in a variety of ways. On the other hand, the clones obtained are thawed directly onto the filter and the released DNA is tested for hybridization with the radioisotope-labeled oligonucleotide used. On the other hand, the obtained clones were cultured individually or in a pool of 10 clones, and the plasmid/nismid D contained therein was cultured.
Isolate NA. Next, this DNA is dropped onto a filter or transferred to the filter by Southern blotting after agarose gel electrophoresis, which can be performed sequentially after digestion with restriction enzymes. The DNA immobilized on the filter is hybridized with radiolabeled oligonucleotides. After autoradiography, blackening of the film indicates where the oligonucleotide has bound to the filter. If the corresponding cosmid or plasmid DNA is derived from 10 clone boules, the related clones are processed one by one to generate the cosmid or plasmid DNA.
hybridize with oligonucleotides one by one. The cosmid or plasmid DNA from clones that tested positive was then cut into fragments by digestion with various restriction enzymes, and the latter were then digested with I
Hybridize with the II secondary oligonucleotide. In this way, it is possible to position the DNA region that hybridizes with the oligonucleotide.

This DNA region can be isolated by subcloning into a sequencing vector (e.g., the sequencing vector pUC8 or a sequencing vector derived therefrom) such as S V B 26, followed by sequencing and comparison with a predetermined amino acid sequence. enabled DN
It is possible to show that A is actually the DNA of interest. Using computer programs it is possible to define the structural genes and the 5' and 3' regions (Tables 1 and 2). Sequence the 1-2 kb A v a I fragment encoding lysozyme from the vector pUC8 or l) S V B 26 to the Streptomyces vector, e.g. pER1!5 (Deutsch Samm under the terms of the Budapest Treaty).
lung von M+kroorganismen u
and Zellkulturen (German Hui Biology and Cell Culture Collection) as DSM4913 in the lysozyme deletion mutant). Lysozyme producing strains, e.g. S. coelico
After transformation into DSM 3030, the yield can be increased 3-5 times.

For cloning in Streptomyces, S.
1ividans is the strain to be tried first as a host organism because it grows relatively easily, and it is expected that the desired gene will be expressed in this Streptomyces.

To construct a gene bank, both the isolated total DNA and the 'a-enriched fragment from the agarose gel were ligated into an E. coli/Streptomyces shuttle vector or a Streptomyces vector. However, even after testing more than 100,000 transformants, it was not possible to identify an S. l1vidans clone containing the lysozyme gene.

However, genes are homologous
It is possible to clone into a stem). Lysozyme genes according to the invention can therefore be identified by complementation of lysozyme deletion mutants.

Showing significantly reduced or no lysozyme production ability,
Stable mutants of S. coelicolor DSM 3030 may occur naturally or be obtained by chemical or physical mutagenesis of spores, mycelial fragments or isolated protoplasts. , coelicolor DSM 3030, using a chemical mutagen, e.g.
Nutyl-N'-nitro-N-nitrosoguanidine (MN
NC), treated with nitrosomethylurea, methylmethanesulfonate, ethylmethanesulfonate, 4-nitroquinoline 1-oxide, hydroxylamine, 5-bromouracil, sodium nitrite, mitomycin C, ethidium bromide or acriflavine or reaction with 8-methoxylpropylene followed by irradiation with long wavelength UV light (365 nm) is preferred. Alternatively, the spores can be exposed to short wavelength UV light (2
54 nm). Treatment with a chemical mutagen, preferably N M N G, or UV light (254 nm) irradiation reduces the survival rate from 0.1% to 3
This is done so that the amount is 0%, preferably 1% to 2%. Stable lysozyme deletion mutant S, coe
Licolor is based on the terms of the Budapest Treaty.
Deutsche Sammlung von Mikr
oorganismen und Zellkultu
It has been deposited with the deposit number DSM4913 under the name DSM4913.

It is known that the protoplast formation and transformation conditions developed for Streptomuces 1ividar+s do not yield successful results in all Streptomyces. Wild type S.

coelicolor Mueller DSM 30
In the case of 30, sufficient transformants were transferred to S, l1vid
The method described for ans (C, J, Thom
pson et al.: (19B2) J, Bacterio
l.

+51.668-677).

In contrast, this method uses the lysozyme deletion mutant S, coelicolor Mueller DSM
It is not possible to obtain transformants of 4913. However, transformation of plasmid DNA is difficult for mutant DS
This can be carried out efficiently using M4913 under the conditions described below.

Mutant DSM4913 was treated with about 17 g/liter peptone from casein, about 3 g/liter peptone from soybean meal, about 2.5 g/liter glucose,
Approximately 5 g/liter of sodium chloride and approximately 2.5 g/liter of sodium chloride
The cells were precultured in a Ca5o medium consisting of 1 liter of hydrogen phosphate and dipotassium, and then transferred to a Ca5o medium enriched with about 5 g/liter of glycine, and kept at 30°C for about 45 hours until reaching the growth stage. Shake. Optimal times and temperatures for growth can be easily established for each mutant by determining the yield of transformants obtained in each case.

Protoplast formation is 0.2! 5 mg/ml ~
1, Omg/ml, preferably 1 mg/ml egg lysozyme, in P buffer [Hopwood DA et al.: rGeniticManipu! ation of
Streptomyces: A Laborato
ryManual J, The John 1nne
s FoundationSNorwich.

England, P, 245 (1985) for 30 to 60 minutes, preferably 40 minutes at room temperature with gentle shaking. For transformation, plasmid DNA encoding thiostrepton resistance and T buffer (
)1opwood et al.: supra, L2413) were mixed with the protoplasts and then modified R3 soft agar containing agar instead of low melting agarose (Shi rahama et al.: 1981, Agric
, Biol.

Chem, 45.1271-1273). The embedded protoplasts are regenerated.
tion) medium, preferably R2YE agar plates (H
(see opwood et al., supra, p. 236).
, lift it up and dry it uncovered in the center of a sterile laboratory bench for 2 to 6 hours, preferably 411 minutes. 20~ at 30℃
After incubation for 26 hours, preferably 24 hours, NB soft agar containing 200 μ3/ml thiostrepton (approximately 3 g/liter Bact beef extract, approximately 5 g
6 g/liter of agar) and further incubated at 30° C. for 6 days.

To create a gene bank, lysozyme-producing strains,
Preferably S, coelicolor DSM 30
The total DNA of 30 is isolated and cut incompletely with a restriction endonuclease, such as 5au3A.

We isolated a 5au3A fragment with a size of 2.8-15 kb and expressed it as S, coelicolor DSM.
3030, linearized and dephosphorylated vector pEB15. Vector pE
B15 is S, venezuelae DSM 40
Plasmid pSV H1 (EP
0070522) Digestion of pEB 15 derived from D. can be performed, for example, with the restriction enzyme Bgtn.

Instead, S, coelicolor DSM 30
30 or other producing strains of this Streptomyces.
A is completely cleaved with, for example, the restriction enzyme SmaI. By DNA-DNA hybridization with the above oligonucleotide probe suitable for the lysozyme gene, S. coelicolor DSM 3
SmaI fragment of total DNA of 030 (approximately 3.3k
It is possible to identify the size of b). The identified fragments were then subjected to E. coli DNA polymerase (
The vector is ligated into the linearized dephosphorylated vector I) 0M4 (EP 025741?) whose ends have been previously blunt-ended by filling in with the Freneau fragment). Cleavage of pGM4 can be performed with the restriction enzyme BamHI.

After treatment with T4 DNA ligase, DNA 'I
The protoplasts of the lysozyme deletion mutant DSM4913 are transformed with the H compound. The resulting clone,
A 5M medium (pH 7, 5) on an agar block at 30°C for 5-7 days. The lysozyme-producing clones were then incubated with heat-inactivated Micrococcus luteus.
Lysozyme test using 1 uteus) cells is identified by the formation of a suspended zone of lysis on an agar plate.

As a result of isolation of plasmids pcell and pce12 from lysozyme-producing clones and de novo transformation with the lysozyme deletion mutant DSM4913, lysozyme-producing transformants are obtained in all cases tested. In contrast, plasmids pcall and pce
After transforming S. 1ividans,
Lysozyme production cannot be detected by agar diffusion test or photometric measurements using Micrococcus luteus.

In contrast, plasmid pcell or 1) CE
S by 12, coelicolor DSM 3
Transformation of 030 or lysozyme overproducing mutants or variants derived therefrom results in a 2-3 fold increase in lysozyme yield compared to the starting strain. Surprisingly, plasmid pce12 contains S,
It was proven to be very stable in coelieolor DSM 3030.

Cloning of the 1.2 kb AvaI fragment of pce12 into the vector pEB 15, which had been cut with BglII, resulted in the J:? of the lysozyme gene. g, in addition to -, S, antibiotics [Berna
n et al.: Gene37.101-110 (1985)
] Plasmid pce13 having the promoter region of the tyrosinase gene is obtained. In the case of pCE 13, S.

The lysozyme production of coelicolor DSM 3030 is increased 3-5 times.

The present invention allows bacterial lysozyme to be produced economically in large quantities, thus making it possible to establish its use as a food preservative or other pharmaceutical applications. Enzymes are particularly advantageous for use in food products because of their optimum pH, which is in the slightly acidic range.

Example 1 Cleavage of DNA with Restriction Endonuclease A single digestion with a suitable enzyme is usually carried out at a total volume of 30 to 11 ml and the reaction temperature is the optimal temperature for each enzyme, usually 37°C. Buffers that can be used include those described by Manatis et al.
atis et al.: Molecular Clonin
g+AL Laboratory Manual, Co1
d Spring Harbor, P, 104 et seq.
Low, medium, and high salt concentrations as described in 1982)
or use buffers specified and recommended by individual manufacturers. mixture? Approximately 1 to 3 per 171g of DNA during the night
U enzyme is used. The incubation time depends on the heat buildup of the enzyme used.
Depending on the time, one hour to several hours may be used. In the case of double digestion with two different enzymes, it is usually possible to add both enzymes to the mixture at the same time. If the salt requirements of the enzymes are significantly different, use the enzyme with a low salt requirement first, increase the salt concentration, and then add the second enzyme.

Completion of digestion can be checked by gel electrophoresis of a portion of the reaction mixture.

Example 2 Phosphatase reaction TE buffer? ? Z (10mM) Lis-HCl, 1mM EDT
Linearized vector DNA with a protruding 5' end, previously dissolved in pH 8.0), was added to a 5 μm 2
0XRAP buffer (800% Tris-HCl, 100mM
Adjust the volume to IL 10071I with MgCI 2.2mM ZnCl2, pH 8,0) and water, then add approximately 30U
of calf intestinal phosphatase (CCI P) (manufactured by Boehringer Mannheim).

After incubation at 37°C for 2 to 3 hours, 5 μm of 10% SDS was added, followed by heating at 68°C for 15 minutes.

After three times of phenol treatment, ethanol precipitation, 70%
Wash with ethanol and place in fresh TE buffer. Another possible method is to add 1 μm CIP to the mixture immediately after restriction enzyme digestion and incubate for an additional 20 minutes at 37°C. Then 2 μm of 0.5M EDT
Add A and heat the mixture at 65°C for 1 hour. Subsequent phenol treatment and precipitation are carried out as described above.

Example 3 Ligase Reaction The ligation of isolated DNA fragments with plasmids or Cosmi1-vectors was carried out by Maniatis et al.
iatis et al.: supra, p. 286 et seq. and p. 474.
, under conditions recommended by ). reaction,
DNA concentration 100-4007J with a volume of 10-20μm
g/ml. Precipitating together the DNA to be ligated and resuspending it in a calculated amount of gauze buffer
It is advantageous for poison. In the case of ligation with plasmids, the ratio of vector plasmid to insert is usually between 1:1 and 1:5, and vice versa in the case of ligation with cosmids.
: Between 1 and 10: ]. The enzyme used is T4
．． DNA ligase (Boehringer Mannheim,
New England Biolabs) 1oo- per mixed solution
Used at a concentration of 4000. Incubation is 14
-18°C for at least 12 hours. To check the reaction, a portion of the reaction mixture, e.g.
In the case of a .mu.m mixture, 3 .mu.m is removed from the reaction vessel and compared on a 0.8% agarose gel with a sample removed from the reaction mixture and stored at 4.degree. C. before adding T4 DNA ligase.

Example 4 Packaging extr.
The packaging extract required for the in vitro packaging of DNA into the heads of phages was prepared using the method of Hohn (
Hohn, B: Wu, R, FJT Collection, Recom
binantDNA, Methods in Enz
ymology, Vol'6B, Academic
Press, New York, 299-309.1
979). Escherichia coli BH32688 strain (
Freeze thaw Iysate,
FTL)) and B HB2690 (sonic extract, 5E) were extracted from glycerol cultures in L broth medium (10 g Bactodributon, 5 g yeast extract, 5 g NaCl, pH 7,5 per liter). ) (50 μm glycerol culture for 5 ml medium). Streak cultures grown at 32°C onto L broth plates and re-incubate at 32°C.
Incubate at °C. Pick 5 colonies from each plate of growth and transfer to two broth plates. One of them is incubated at 32°C and the other at 42°C. Only single colonies that grow at 32°C and do not grow at 42°C are used in the next step.

In each case, one of the colonies showing no growth at 42°C is now streaked onto an L-broth plate and incubated overnight at 32°C. To prepare packaging extracts, one small colony is selected from each of these plates and cultured in 10 ml of Shigros medium. Cultures are grown overnight at 32°C and used the next day to inoculate the main culture.

To prepare FTL extracts, 800 ml of fresh broth medium was inoculated with 10% BH32688 preculture and incubated at OD 600 at 220 rpm on a shaker at 32 °C.
Culture until the value reaches 0.45 to 0.50.

The culture is then heated to 42-43° C. with rotation over the flame of a Bunsen burner. The temperature is measured nonsterilely by inserting a thermometer directly into the suspension. Incubation is then carried out in a rotating water bath for 15 minutes at 42-44°C with gentle shaking, then for 2.5 hours at 37°C with vigorous shaking. The suspension was cooled to 10°C in ice water and then 200ml was added to 6. 4 at OOOrpm
Centrifuge in 5 orvall RC-5 for 10 minutes at °C. Sprinkle each pellet with 0.3 ml of cold sucrose solution (1
0% sucrose dissolved in 50mM Tris, p)I
7.4) and mix it without creating air bubbles, and
1. Transfer to a centrifuge tube and stir with 30 μl of fresh lysozyme solution (2 mg/ml aqueous solution). 6. Freeze the mixture in dry ice. After at least 30 minutes, slowly thaw the centrifuge tube to room temperature. After completely melting, 250μ
m water-cooled Ml buffer (410 μm H2O, 1 μm β
- Mercaptoethanol, 6μm 0.5M Tris-HCl, pH 7,4,300) tl 50mM spermidine, 9μm IMMgClz, 75μm 0.1M A
TP) to the contents of each tube and, after careful mixing,
Centrifuge for 25 min at 2 °C at 5,000 rpm (
Beckman L8-M (30 Ultracentrifuge 11).

The supernatants are then combined and 25711 portions are frozen in dry ice and stored at -86°C until use.

To prepare the SE packaging extract, 400 m
1 of the broth medium is inoculated with 4 ml of an overnight culture of strain BH32690, and then the steps are carried out exactly as described for strain BH82688, up to centrifugation in a 5-orvall centrifuge. BHB obtained in two centrifuge tubes
Each pellet of 2690 strains was mixed with 3 ml of cold buffer A (
20mM) Lis-HCI (pH 8,0), 3mM MgC
12, 10mM β-mercaptoethanol, 1mM E
Stir in D T A) without forming air bubbles, and pour the suspension into a clear plastic container. Place the plastic tube in the salt/ice/water mixture and cycle for 5 seconds (III pulses) at maximum power.
) and sonicate with an ultrasonic device (Branson sonicator). Check the viscosity of the solution with a Pasteur pipette between cycles. A large increase in solution viscosity is observed after the first few cycles (release of high molecular weight DN from the cells), then the viscosity decreases with increasing number of cycles (DNA fragments into low molecular weight fragments). ). During each cycle, the suspension is repeatedly cooled sufficiently so that the temperature of the suspension never exceeds 20°C. Ten cycles are required to bring the sonicated suspension to a low viscosity. After sonication, the suspension was heated at 6,000 rpm.
Centrifuge for 10 min at 4°C.
5 refrigerated centrifuge, 5S-340-ter), aspirate the supernatant from the small pellet. Add 0.6 ml of cold M1 buffer (see above) to all supernatants of each 200111, mix, and then transfer 20 ml of each to 0.5 ml Eppendorf reaction tubes that have been pre-chilled on dry ice. Transfer.

Store the aliquoted samples at -86°C.

Alternatively, various manufacturers, such as Boehringer Mannheim and Amersham Buchle
It is also possible to purchase ready-made packaged extracts, such as from R. Braunschweig.

Example 5 Packaging in human phages 3 μm of the glue gauze mixture described in Example 3 was stirred with 9 μm of SE extract (Example 4) in an ice water bath and after 2 minutes 10 μm
Add m FTLh11 product (Example 4). Then incubate for 10 minutes at room temperature.

Then 500 μM of 3M buffer (100mM NaCl
, 10mM Mg5OIL, 50mM) Squirrel MCI, pH
7-Add 5,0,01% gelatin). This mixture can be used for transduction reactions.

Example 6 Transduction of E. coli ED8767 50 μm of E. coli ED8767 was added to 5 ml of broth medium supplemented with 0.4% maltose and 10 mM Mg5
Inoculate a well-grown liquid culture of strain 767. Bacteria are shaken at 37° C. for approximately 12 hours and harvested in a tabletop centrifuge after reaching an early stationary phase.

The precipitate was added to 0.5 ml of buffer (10 mM MgSOIL, 0
．． 4% maltose) and resuspend in 0.25 ml of the same buffer. Cosmidov 7- obtained in Example 5
10 μm of this concentrated bacterial suspension
Add to. This is incubated at 37°C for 15-30 minutes. Then add 0.5 ml of broth medium,
Incubate the mixture again at 37°C for 30-60 minutes. 100 μm of the mixture is then spread onto a broth plate containing 15 μg/ml ampicillin. Incubate the plate at 37°C until well-grown colonies are obtained.

Example 7 Partial digestion of DNA Partial digestion of total DNA from Streptomyces was published in the magazine “Focus J (BethesdaR)”.
esearch Laboratories, Vol.
, 7, No. 2, p. 1985.3). Streptomyces S. coelicolo
r The total DNA isolated from the DSM 3030 strain was cut into fragments of 30-40 kb with the restriction enzyme 5alI (T30-40 kb). 50 μm DNA solution, 40111 (7)
TE buffer (10mM) Lis-f (C1, 1mM EDT
AS pH 8.0), a total volume 100 μm mixture containing 10 μm of IOX high salt Takashi buffer and 10 TJJ of 5alI has proven particularly suitable. The mixture is incubated at 37°C for 10 minutes and then immediately transferred to ice. After extraction with phenol/chloroborum, D
The NA is precipitated with ethanol, washed with 70% ethanol, and then resuspended in 50 ml of TE buffer.

In this method, the 5ail enzyme does not cut all possible cleavage sites.

Example 8 S in E. coli, coelicolor DSM
Creation of a cosmid gene bank of 3030 DNA Total DNA was extracted from Streptomyces S. coelical
orDSM strain 3030 and isolated by the method described by A Hopwood et al. Partially digested with 5all restriction enzymes in the following manner. A portion of the digested Streptomyces DNA was digested with restriction enzyme gsa as described in Example 3.
The cosmid vector 1) is ligated with HC79 (see Example 2), which has been completely cleaved with Il and then dephosphorylated. A 20 μm ligase mixture of 3111 is packaged into the head of λ phage (Examples 4-6). The resulting recombinant cosmid vector is used to infect E. coli strain ED8767. Recombinant bacterial colonies are sacrificed on broth plates supplemented with 50 μm/ml ampicillin (Arnp). Approximately 2,000 colonies are picked and transferred into the grid of a fresh broth/Amp plate so that individual clones can be easily found again. A copy of each plate (d
upl 1cate). To check whether the gene bank is complete, a single colony is m1
The cosmid DNA that is isolated by incorporating into the former lysis is completely cleaved with the enzyme 5alI. The resulting fragment pattern shows cosmid DNA with a size of 30-40 kb.
is included in all clones.

To search for the lysozyme gene in the gene bank, in each case 10 colonies taken in the basal order were divided into 5
Co-inoculate 5 ml of broth medium containing 0 μg'/ml ampicillin and shake overnight at 37°C. )
mlnilyses are carried out on the relevant suspensions, and the resulting mixture of 10 cosmid DNAs in each case is completely digested with the restriction enzyme 5alI. Fragments are fractionated on a 0.8% agarose gel and transferred to nylon filters by Southern blotting. Streptomyces S. coeli
5al I digested total DNA in color DSM3030 is also applied to the same gel as a control. After fixing the DNA on the filter, the latter is used directly in the next hybridization reaction.

These filters were prepared as described in Example 13 by 1.
Hybridize with the following free-labeled oligonucleotide probe.

5'-TTCGC(G/C)TACATCAAG GC
(G/C) AC (G/C) GAG GG (G/C)
AC (G/C) AACTACAA-3' filter was hybridized at 55-60°C, then 60-6
Wash at 5°C. The cosmid DNAs of approximately 2,000 cosmid clones obtained are pooled in groups of 10 and reacted with a radiolabeled mixture of oligonucleotides having the above sequence.

After autoradiography, the position on the filter is S.
, coelicolor DSM 3030 full DN
1 each, indicating a signal that is identical to the signal from A
It was possible to find a total of 11 pools with 0 clones. Cosmid DNA was isolated by inoculating related clones from 10 pools one by one. Hybridization with previously used oligonucleotides is indicated by dots, thereby identifying clones corresponding to positive signals in each of the 10 pools.

Example 9 Subcloning DNA fragments from cosmid clones Cosmid DNA from one of the positive cosmid vectors was
Purify on a sCl/EtBr gradient and digest the isolated DNA to completion with restriction enzyme 5ail. The resulting DNA fragment was similarly digested with 5all.
) and then treated with alkaline phosphatase (calf intestinal phosphatase, Boehringer Mannheim, see Example 2).
Concatenate with. pSVB26 is a known vector p
U C8 [Vieira and Messing: G
ene 19 (1982) 259-68, and differs only in the positional direction and nature of the restriction enzyme cleavage site in the polylinker. It is likewise possible to use pUC8 for the cloning described below.

After transformation of competent cells of the large 11i1 mHB 101 strain, selection of clones containing plasmid DNA is carried out on broth medium containing 5071 g/ml ampicillin. After colony growth, colony hybridization (Example 15) is used to determine which clones contain the fragment of interest. Plasmid D
NA was extracted from clones showing positive reaction.
The clones are isolated by S and examined by 5alI digestion to see if the clones contain the desired 5aLI fragment.

The 5alI fragment is finally identified by hybridization with the oligonucleotide used for screening.

It is also possible to subclon in a similar manner other restriction fragments 1 from cosmid clones that react positively with the oligonucleotide probe in vector pSV826. For example, an approximately 1.2 kb sized AvaI fragment from a cosmid clone was identified, the overhanging ends were filled in with Klenow bolimerase, and after isolation from an agarose gel, the SmaI fragment of pSV826 was identified.
Cloned into the cleavage site. Two different Bs5H ■
Fragments are similarly identified using different oligonucleotides to fill in the ends and, after isolation from an agarose gel, are cloned into the single Smal cleavage site of pSVB26. In this way, various restriction fragments can be cloned into pSVB26.

As a result of restriction enzyme mapping of these fragments, the restriction enzyme map shown in FIG. 1 was obtained, which includes the lysozyme gene and surrounding regions.

Since this region contains a unique restriction cleavage site, specific deletion is performed by double digestion with an appropriate enzyme and an enzyme that recognizes one of the restriction cleavage sites in the polynoncar region of pSVB26. and use the latter for subsequent sequencing. Since psVB26 differs from the pUC vector only in the polylinker sequence, a normal pUC primer is used for sequencing the 5alI fragment by the Sanger method. moreover,
The oligonucleotide used for screening is used as a primer. The various constructs used for sequencing are shown again in more detail in FIG. 2. The sequence of the 1 to 2 kb S maI/AvaI fragment shown in FIG. 2 is shown in Table 1, and the sequence of the lysozyme structural gene is shown in Table 2.

Example 10 Identification of hybridizing fragments from total DNA Total DNA isolated from coelicolor was treated with various restriction enzymes, such as 5alI, Bs5HI, Sm
aI, Aval, BglII, BamHI, EcoRI
, PvuII, etc., each singly and completely digested. The resulting fragment mixture is fractionated on a 0.8% agarose gel and transferred to nylon filters by Southern blotting. The filters are treated as described in Example 13 to hybridize with radiolabeled oligonucleotides. The optimal hybridization temperature is determined by varying the hybridization and wash temperatures from 30-60°C.

Visualize hybridization by autoradiography. Total DNA fractionated into unique fragments at the highest possible hybridization and washing temperatures
The temperature that can be identified from A is thus searched for each oligonucleotide (mixture). In particular, the sequence 5'-TTCGC with a length of 38 nucleotides
(G/C)TACATCAAG GC(G/C) AC
(G/C) GAG GG (G/C) AC (G/c)
Good results are achieved with oligonucleotide mixtures having AACTACAA-3'. Shorter oligonucleotide mixtures do not give satisfactory results.

S at a hybridization and washing temperature of 1.60-65°C using an oligonucleotide mixture of 38 nucleotides in length.

coelicolor DSM 3030 Once a clear signal is obtained from total DNA, for example, a Small fragment of about 3.3 kb, a 5alI fragment of about 1.3 kb, and an Ava of about 1.2 kb.
Bs5H fragment and approximately 0.8 kb in size.
I[S] fragment thus using this oligonucleotide mixture.

Identified from the total DNA of coelicolor DSM 3030.

Digestion with EcoRI, BamHI, Bgll, XhoI, etc. allows only very large fragments to be observed.

Example 1] S, coelicolor DSM 3030 full DN
Cloning of the 5alI fragment from A By the steps described in Example 10, it was possible to identify a 5alI fragment with a size around 1.3 kb that showed a positive signal with the oligonucleotide mixture used to search for the lysozyme gene. Ta. S, coeli
The total DNA from color DSM 3030 was completely digested with the restriction enzyme 5alI and fragments of size 1.1-1.5 kb were isolated from an agarose gel by the steps described in Example 21. 5 purified fragments
After complete digestion with alI, alkaline phosphatase (calf intestinal phosphatase, Boehringer
Mannheim)-treated vector pSVB2
Connect with 6. Competent cells of E. coli strain H8101 were transformed with the ligase mixture to
) Select clones containing the plasmid on L broth medium containing i g/ml ampicillin. Approximately 1,000 colonies obtained are picked up as substrates and duplicates are made for each plate. In each case, 10 clones are transferred together into 5 ml of broth medium and after propagation the plasmid DNA is isolated by m1nilysis and subjected to an agarose gel undigested. After a brief electrophoresis, the DNA is transferred to a nylon filter by Southern blotting. These filters are then hybridized with the oligonucleotide mixture described above under pre-established optimal conditions. The positive pool was found in this way.

The above steps are performed by inoculating the clones represented in the pool one by one. This makes it possible to unambiguously identify clones showing a positive reaction in each case. The plasmids isolated from these single clones all contained the same 5alI fragment. The subsequent steps are carried out as described in Example 9.

Example 12 Southern DNA migration DNA fragments are fractionated by 0.8% agarose gel electrophoresis in the presence of standard DNA. After the electrophoresis is completed, transfer the gel to a plastic container and
M hydrochloric acid for 15-20 min, then denaturing buffer (0,5
MNaOH11,5MNaC1) for 2 x 30 min and finally neutralization buffer ji (IM) Lis-HC1 (pH 8,0
), 1.5 M NaC1) for 2 x 30 min in the hanging device (nu
tatingdevice). In each case, the buffer solution is added so that the gel is sufficiently covered.

The gel was then coated with two layers of absorbent paper (Whatman, 3MM
) on page 385 of Maniatis et al. (see above).

However, a nylon filter (Genescreen
Plus, manufactured by NEN Corporation) is used instead of the nitrocellulose filter. Plotting is carried out for at least 16 hours starting with absorbent paper. After the transfer is complete, mark the gel slot and insert the nylon filter into the
Place in the oven at 5℃. Then the filter is 10X
Adsorption paper (Whatman,
3MM) and irradiated with short wavelength UV light for 2 minutes (UV lamp, Desaga Minuvis
, Wavelength: 254 μm, Filter/UV light source distance if:
10-12cm). During this time, keep the side of the filter containing the DNA away from the UV light source. After irradiation, air dry the filter.

EXAMPLE 13 Hybridization of DNA bound to a filter is carried out substantially by Gibco
Magazine published by BRL (rfocusJ, Vol. 9)
, NO62, 1987, p. 1-2).

Pre-hybridize the filter with hybridization buffer (5xs
sc, 20 resistant monosodium phosphate (pH 7,0), 10
X Denhardt, 7% SDS, 100 μg/ml salmon sperm DNA denatured by boiling for 15 minutes)
Wash at 65°C for at least 2 hours. Dextran 5R acid solution (50%) was then added to a final concentration of 10%, and the radioactively labeled oligonucleotides were added to
Add to the hybridization solution at a concentration of 5 ng/ml.

Incubation is then carried out for at least 16 hours with gentle shaking in a shaking incubator. Although the individual incubation temperature varies depending on the oligonucleotide, it should be optimized in advance. Buffer for cleaning filters? &I
(3X SSC 110mM sodium phosphate, (pH 7,0
), 10X Denhardt's, 5% 5DS). After shaking for 1 hour at hybridization temperature, Wash Buffer I was replaced with Wash Buffer II (IXSSC
51% 5DS), the latter being replaced after 30 minutes. After 30 minutes, expose the wet filter in fresh wash buffer 1 to an XHg optical film (eg, Kodak X51) in a cassette. Exposure time is between 4 hours and 5 days at 80°C.

Example 14 Autoradiography Hybridized Filters
Fix it on M paper. Exposure was carried out at -80°C using an intensifying screen in a light-tight cassette using a suitable X-ray film (Kodak X-OmatAR, Kodak XS 1).
Do it with The film is then developed.

Example 15 Colony Hybridization Plaque/Colony Screen Filter (Co1on
y/Plaque Screen (registered trademark), NENRe
Manufactured by Search Products, Catalog No.
NEF-978/978A) is placed on the agar plate containing the colonies. The filter was removed after 2-3 minutes and first soaked in 10% SDS with the bacterial colonies attached to the surface.
Leave it on the paper for 5 minutes. Then, the filter was preliminarily 0.5
MN aOHll, Wha soaked in 5M NaCl
Transfer to tmann a M M paper for 5 minutes. 0.5M in advance
) Lis-HCl (pH 8,0), 1.5M Nacl
Transfer to Whatmann 3MM paper soaked in water for 5 minutes and finally to dry Whatmann 3MM paper. After drying, gently loosen the ζ filter to remove adhered colony residue from the filter. Revised night (3XSSC5
0.1% 5DS) at 65° C. for several hours.

Example 1G Oligonucleotide Synthesis The synthesis of the NA building blocks is illustrated using the oligonucleotide from Example 10 as an example. For solid phase synthesis, 3
The nucleoside located at the 'terminus, ie adenine in this case, is covalently linked to the carrier via the 3' hydroxyl group. The carrier material is CPG (:JI-ordered perforated glass) having long-chain aminoalkyl groups as functional groups.
1 led poreglass). In the next synthesis step, the base component is used as 5-0-dimethoxytrityl nucleoside-3-phosphate β-cyanoethyl dialkylamide. Here, adenine is present as an N6-benzoyl compound, cytosine is present as an N4-benzoyl compound, guanine is present as an N2-isobutyl compound, and thymine is present without a protecting group. The following agent is treated sequentially with 25 mg of polymeric carrier containing 0.2 μmol of bound 5-0-dimethoxytonothyl-N6-penzoyl-2-deo, oxyadenosine.

A) Acetonitrile B) 3% trichloroacetic acid in dichloromethane C)
acetonitrile D) 5 μmol of the appropriate nucleoside-3-0-phosphite and 25 μnot tetrazole (0,1
5 ml of anhydrous acetonitrile solution) E) Acetonitrile F) 20% acetic anhydride in tetrahydrofuran containing 40% lutidine and 10% dimethylaminopyridine G) Acetonitrile ■) Lutidine/water/5:4:1 by volume 3% iodine dissolved in a mixture of tetrahydrofuran Here, "phosphite" is 2-thiokyribose-3-monophosphoric acid mono-
β-cyanoethyl ester is used, and the third party is saturated with a diisopropylamino group. The yield of each individual synthetic step is determined by detrichation (det.
ritylation) can be determined by reaction B) by measuring the absorption of the dimethoxytrityl cation at a wavelength of 496 nm in a spectrophotometer. After the synthesis is complete, the dimethoxytrityl group is removed as described under A) to C). Ammonia treatment releases the oligonucleotide from the carrier and simultaneously removes the β-cyanoethyl group. The amino protecting group is quantitatively removed from the base by treating the oligomer with concentrated ammonia for 16 hours at 50°C.

The crude product thus obtained is purified by polyacrylamide gel electrophoresis.

Example 17 Protein Sequence Analysis Sequence analysis of HPLC-purified proteins or protein fragments was performed using a protein sequencer (App
477A) manufactured by Biosystems.

Example 18 DNA Sequence Analysis DNA sequence analysis was performed by Sanger et al. (Proc.
tl, Acad, Sci, USA 74.54
63-5467.1977) for double-stranded plasmid DNA.
ation process), He1nrich (
Guidelines for quick and
simple PlasmidSequence! n
It was carried out using the declination method according to G. G., Boehringer Mannheim, 1986). The “normal” or “reverse” vector used for sequencing from the UC vector
ed) J Brimer can also be used for sequencing from pSVB vectors. DNA used for sequencing is purified twice by CsC1/EtBr gradients.

Sequencing is carried out using reagents that are commercially available as kits, such as those by Boehringer Mannheim (Talenow Polymerase).

Example 19 Sequencing Gel Sequenced samples are fractionated on a 6% polyacrylamide/urea gel. To compress the broadly separated bands in the lower part of the gel, and to achieve the maximum possible resolution in the upper part, the cathode vessel was filled with a running buffer containing 1M sodium acetate. Use gradient.

Example 20 Radioisotope Labeling of Oligonucleotides The step of radiolabeling purified oligonucleotides consists essentially of Gi
beo BRL Publishing Magazine "Forgus"(＼'o
1.9, No. 092.1987.1). 2 (Purified desalted oligonucleotide with absorbance 1 at 30 nm is placed in 40 μm of double distilled water and 1 μm of this is used for standard labeling reactions.

15-2011+ used radioactivity (γ-32p
-ATP, 6, 0OOCi/mmo I, New
(manufactured by England Nuclear) and concentrated (
Speed Vac condenser (Bachhofer), 7 μm double distilled water. 1μm 10x kinase buffer (0,5M) lis-ci (pH 7,6),
111
1 re-g4 oligonucleotide and 1 μm T4
Add polynucleotide kinase (approximately 7 units).

The mixture is incubated at 37° C. for approximately 30 minutes and unreacted ATP is removed with a Sephadex 015 column.

The radiolabeled oligonucleotide then appears clearly in front of the major amount of unreacted ATP as the first fraction, which can be measured directly by a handheld counter. The purified oligonucleotides are used directly in subsequent hybridization experiments.

Example 21 Isolation of DNA Fragments from Agarose Gels After fractionating DNA by gel electrophoresis in a horizontal apparatus using sodium acetate as the running buffer overnight, the DNA fragments of interest were located using long wavelength IJV light (366 nm). )
Determine by comparing with the marker fragment under. The gel in the band width region is cut out to a thickness of about 1 to 2 mm, and the gel is returned to the device and filled with a running buffer. Run the electrophoresis again for a very short time (45-60 seconds) under normal conditions, remove the buffer from the cut out spot with a pipette, and repeat this operation until the desired DNA band completely disappears from the gel. . The buffers removed from the gel slots are combined and extracted with phenol/chloroform, and the eluted DNA is resuspended as usual after TE mild buffer ethanol precipitation.

Example 22 Selection of oligonucleotide sequence The following amino acid sequence is the N of lysozyme protein.
obtained by sequencing the termini and B rCN fragments.

N-terminal sequence: Arg Trp 1e -Ile-Asn-Trp B rCN fragment 2: 5p Thr-Glu- to 1-Thr-Asn--AsrI-
Lays-TieSer-Phe-Ala-Tyr-1
1e-Lys-Ala-rla-Ala to ly-Tyr
-Agp-Tyr-Phe-X -Aspla Set Ile ^rg-Phe--Ala--Tyr Val Asn B rCN fragment 3: Alt-Ala-Lys-X -! 'ro-Phe-T
rp-Val-Δ1a-Fiis-τrp-G1. y-
Val-5er-Ala-PNi5er-Gly-Pha-Pro-Thr The following oligonucleotide mixture is synthesized from the amino acid sequences indicated in highlighted letters.

N-terminal sequence: G; GG/CGTG/CCAA/GG
GG/CATT/CGAT/CGT or CAA/G for underlined sequences
GGN ATT/CGAT/CGTB rCN fragment 3: G to TTT/CT CG/CCAT/CT to GTG/C
GG GG or single oligonucleotide TTCTGG GTG GCG CACTGG GGT
TCTGG GTCGCCAC1'GG GGTTC
TGG GTG GCCCACTGG GGTTCTG
G GTCGCG CACTGG GG For the oligonucleotide described, the producer strain (producer 5
In some cases, it was possible to obtain reproducible signals for hybridization with genomic blots of (train). This is the 14me from the N-terminal region.
Particularly applicable to r and B rCN fragments 3 to 20 mer. However, the clones identified with the corresponding oligonucleotides did not prove to be correct.

The amino acid sequence found from B rCN fragment 2 showed significant differences between experiments. However, the underlined sequences above are from the known fungus Chala
A significantly better match to the sequence from rops is can be shown by computer comparison. The following oligonucleotides from this sequence are synthesized considering only the G and C residues in the third position of the relevant codon.

5-TTCGC(G/C)TACATCAAGGC(
G/C) AC(G/C) GAG GG(G/C) A
Genome D of C (G/C) AACTACAA-3 producing strain
In hybridization with NA, reproducible signals are obtained using this 38mer. Using oligonucleotides, we successfully identified the lysozyme gene from the E. coli gene bank. D of the cloned gene
NA sequencing showed that the asparagine residue identified by amino acid sequencing at position 14 of BrCN fragment 2 was replaced by a lysine residue.

However, since the individual codons differ only in the third position, this error does not adversely affect the binding ability of the oligonucleotides used.

Example 23 S. coelicolor by chemical mutagenesis
To prepare an isolated spore suspension of DSM 3030 lysozyme deletion mutant, lysozyme-producing S.
coelicolor DSM 3030 in SM medium (
Incubate in agar slant tubes containing 20 g/l soybean meal, 20 g/l mannitol, 18 g/l agar, pH 7.5) at 30° C. for at least 7 days until clear sporulation is observed. 5 to which 0.01% (W/V) polyoxyethylene sorbitan monooleate (Tween 80) had been added.
Rinse with 0.9% (w/v) sodium chloride solution in an ultrasonic water bath for 20 seconds. Cell debris is removed by filtration through a sterile membrane filter with pore size 5) tm. 10. The spores contained in the PA solution. ．． 10 in 0OOX g
Centrifuge for minutes to collect. For the next chemical mutation, at least 106 spores were added to 2 ml of 0.9%
v) 8 ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNC; suspended in sodium chloride;
, 3 mg/m of 'rM buffered i& (50 mmol/l Tris-maleic acid, pH 9,0) solution is added and the mixture is incubated for 60 minutes at room temperature protected from light. Under these conditions, a spore survival rate of about 1% is obtained. Spores were pelleted by centrifugation at 10,0 OOX g for 10 min and washed three times with 10 ml of 0.9% (w/v) sodium chloride each time, followed by 5 ml of 0.9% (w/v) sodium chloride each time.
v) Suspend in sodium chloride and dilute appropriately to make SM
Spread onto nutrient medium plates and incubate for at least 5 days at 30°C. The resulting colonies are transferred to 5M agar blocks (0.5 cm diameter) and incubated for 5 days at 30° C. in a humidified cabinet. The blocks were then transferred to plates containing Micrococcus test agar and placed in a humidified cabinet! - Incubate for a further 16 hours at 30°C.

Lysozyme test agar was suspended in O, l molar sodium acetate buffer (pH 5,0) using lyophilized Micr.
ococcus 1uteus ATCC4(398(
Boehringer Mannheim) (0.4mg/ml
). This is Ul t+・atu+・ra
Homogenized by X treatment for 5 min and incubated at 60 °C before use.
0.9% agar was added.

The action of extracellular proteases is demonstrated by the formation of clear zones directly adjacent to the colonies on the micrococcus test agar used. Lysozyme forms another turbid zone adjacent to its outside.

From the 10,000 colonies examined, S. coelicolor DS no longer shows a cloudy melting zone.
One of the M3030 lysothyme negative mutants was isolated. 50 mm of the latter from appropriate 3M nutrient medium plates.
After shaking for 3.5.7.10 days at 30°C, DE3440
Micrococcus 1uteus described in 735
Determined by turbidity measurement using The isolated mutants were tested by repeated shaking culture tests after protoplast formation in SM nutrient solution and vector pcM4 (EP025
741? ) is stable, as shown by transformation.

Example 24 By physical mutagenesis S. coelicolor
Isolation example 2 of lysozyme deletion mutant of 05M4913
3 and at least 106 S.

5m of coelicolor DSM 3030 spores
9% (w/v) sodium chloride and 0.01% (
W/V) A filtered spore suspension was prepared by suspending the spores in polyoxyethylene sorbitan monooleate (Tween 80). The steps used for the following UV mutations were performed using the method described (Hopewood D, A, et al.:
7-38). UV light for this (254nm
The irradiation time was chosen such that the survival rate obtained was 2%. Spreading the irradiated spores at appropriate dilutions onto 3M nutrient medium plates (see Example 23);
Incubate at 30°C for at least 5 days.

The colonies obtained are transferred to 5M agar blocks (0.5 cm diameter) and incubated for 5 days at 30° C. in a humidified cabinet. The flocs are transferred to plates containing Micrococcus test agar (see Example 23) and incubated for approximately 16 hours at 30°C in a humidified cabinet. From 7 000 colonies examined, a lysozyme mutant, S. coelicolor, which does not show a cloudy melting zone on Micrococcus test plates.
DSM 4913 was isolated.

This mutant contained 50 mmol/liter F (EPES
(pH 7,0) in a 5M nutrient solution (see Example 23) after brotoblast formation and vector p CM 4 (EP 02571!).
+7) was proven to be stable even after transformation.

Example 25 Preparation and transformation of protoplasts of S. coelicolor strain The lysozyme-negative mutant S. coelicolor DSM 4913 isolated as in Example 24 was transformed into
ml of Ca5o nutrient solution (17 g/l peptone from casein, 3 g/l peptone from soybean meal, 2.5 g/l glucose, 5 g/l sodium chloride, 2.5 g/l phosphoric acid) (dipotassium hydrogen) at 30°C for 3 days. Transfer 5 ml of this culture to 50 ml of Ca5o nutrient solution supplemented with 5 g/liter glycine and shake at 30° C. for approximately 45 hours until stationary growth is reached.

The cell mycelium was finally fractionated using a glass homogenizer (d
ivide) and centrifuge at 5,000×g for 10 minutes. Transfer the cells to 25 ml of P-buffered m()Iopwo
od O.

et al.: p, 245, see above), 5. Centrifuge at OOO'Xg for 10 minutes, 12.
Place in 5mt P buffer. 12.5ml of 2mg/m
1 of chicken egg lysozyme in P buffer is added and shaken gently at room temperature for about 40 minutes until microscopic examination shows that the mycelium has fully disintegrated into protoplasts. Add 25 ml of P buffer and filter through cotton wool. The filtrate is centrifuged at 3,0 OOX g for 10 minutes, the precipitated Protoplast I. is resuspended in 2 ml of P buffer, divided into 20 OItl portions and frozen at -80°C.

In transformation, 200) tI S,
coelicolor DSM 4]3 protoplasts were incubated with 20 μm of plasmid DNA encoding thiosj. lebuton resistance (corresponding to lng to 1 μg of DNA) and 500/l of T buffer? a (Hopwood
D, A et al.: l), 24G, see above) and immediately plated on 7 R2YE plates (Hop
XJood D, A, et al: p.

236, supra) and dry to 70% of its original weight. It is then layered with 4 ml of modified R3 soft agar pre-warmed to 42°C and dried for 4 hours in the middle of a sterile bench (class 2) without a lid. Modified R3 soft agar [Sl+1raha+na
et al: Ag+'iC, Bial, CI+em
, 45, 1271-1273 (1981)] is
171 g/liter sucrose, Log/liter glucose, 4g/liter bactobebutone, 4
g/l yeast extract, 0.5 g/l potassium chloride, 8.1 g/l magnesium chloride, 2
, 2 g/l calcium chloride, 0.2 g/l potassium dihydrogen phosphate, 4 g/l agar, 25
Contains mmol/liter of TES buffer 7 nights (N-tris[hydroxymethylcoatyl-2-aminoethanesulfonic acid, pH 7,2). The inoculated and dried plates were incubated for 24 hours at 30°C and treated with thiostrepton (200 Izg) dissolved in dimethyl sulfoxide.
2 of NB soft agar (3 g/liter Bactobeef extract, 5 g/liter Bactovep, 6 g/liter agar) pre-warmed to 42°C with the addition of
．． 5. ml and incubate at 30°C for at least 6 hours.
Hate the ink for days.

Example 2G Isolation vector pGM4 (Figure 3) of Hector blasmite was obtained from EP 025741
7, Streptomyces gha
Plasmid ps from S. naensis DSM2932
Prepared from cs and S.

D, A by transforming coelicolor DSM 4913.

HOpwood et al.: (]. 85 (see above)
Isolate as described in .

Vector pEB1!5 (FIG. 4) is S.

Plasmid pS VH1 obtained from Venezuelae DSM 40755 (EP 0070522)
It originates from

2.5 kb of psVHl and 3. The vector p, which encodes thiostrepton resistance with a size of 1.1 kb by removing two 8gln fragments of the size of Okb.
B+ from SLE41 (DE 3412093):
Replace with II fragment. As a result, 8.15k
A vector pEB2 of size b is obtained (Wohl
1eben W, et al. (1985) In: S
ix Int, Symp.

on Actu+mycetes Biology
Szabo G, Biro S,, oodfel
low M, fJW collection 99-101. Akademi
ai Kiado.

Budapest).

4 of pEB2. (3 kb Ss tII fragment encoding neomycin resistance (AP H-I),
Plasmid pL J 68001opwood D,A
et al.: 11.298, see above), 1. O
kb Ss tII fragment. As a result, a 5.7 kb plasmid l) E B3 is obtained. Partial 5au3A digestion of pEB3 then allows l) the minimal replicon of S V H1 to be placed into a 2.4 kb fragment.

5, 1 kb vector plasmid pEB15 (4th
Figure) shows this fragment, which also encodes thiostrepton resistance, p5LE41 (DE 34120
93) and the 1.1 kb Be11 fragment of plasmid p I J 702 [Hopwood D,
A et al: p.

292, supra, Bernon et al.: Gene 37.1
01-110 (1985)] adjacent to a 1.6 kb Bcll fragment encoding thiosinase.

S in vector plasmid pEB15.

coelicolor DSM 4913, S. coelicolor DSM 4913 was transformed.

You can get coelicolor DSM 4914.

S, coelicolor DSM 4914 is De
Sammlung fur Mikro
or3anismen und Zellkultur
It has been deposited in en.

pEB 15, S, coelicolor DSM
4914 to Hopwood D, A et al.: p, 85
It was similarly isolated as described by.

Example 27 S, coelicolor DSM 3030 5a
Cloning of the u3A DNA fragment into the lysozyme deletion mutant DSM4913 Lysozyme-producing S, coelicolor DSM
The total DNA of 3030 was collected by A. Tlopvood et al.
p. 79, supra), incomplete cleavage with restriction endonuclease 5au3A (see Example 7) and fractionation on a 0.7% agarose gel. A .8 kb to 15 kb fragment was isolated (see Example 21).

Vector plasmid isolated as in Example 26;
I) E B 15 restriction endonuclease Bgln
(see Example 1) and dephosphorylated as in Example 2. 0.1 μg S, coelicol
o+° isolated Sa u 3 A fragment and 0
．． The pEB15 DNA cut with BglII at 05713 and dephosphorylated is used in the next gauze reaction (see Example 3).

The method described in Example 25 is then used to transform the bud blasts of the lysozyme deletion mutant isolated as in Example 24.

Example 28 S, coelicolor DSM 3030, 3.
Cloning of the 3 kb SmaI DNA fragment into the lysozyme deletion mutant DSM4913 S. coelicolor DSM 30, which had been identified by a 38 kb oligonucleotide probe specific for the lysozyme gene (see Example 10)
A 3.3 kb Small fragment of 30 total DNA was isolated as described in Example 21.

The vector p0M4 (EP O, 257, 417) was completely cut with the restriction endonuclease BamHI and
Fragment the overhanging ends (Maniatis T, et al.:
p. 113, supra), treated with alkaline phosphatase (see Example 2) to produce S. coelico
The 3.3 kb size isolated SmaI fragment of lorDNA is ligated by T4 DNA ligase (see Example 3). Then coelicolor DSM 4913 with ligase mixture
Protoplus! (see Example 25).

Example 29 Detection of a mutant with complementary lysozyme productivity S obtained as in Example 27 or Example 28.

Coel 1color clones were transferred to a block of 9M nutrient medium supplemented with 30 μg/ml thiostrepton and incubated for 5 days at 30°C in a humidified cabinet, followed by lysozyme test agar (see Example 23). Move it to the top. 1, obtained as in Example 27, after incubation for 16 hours at 30°C in a humidified cabinet.
One of the 500 clones and one of the 150 clones obtained as in Example 28, wild-type S, coel
The production corresponding to lysozyme production of icolor DSM 3030 was clearly demonstrated by the formation of a cloudy melting zone on the Micrococcus test agar.

Example 30 Plasmid isolation and characterization of lysozyme-producing clones Plasmids pcell and pce12 were
It was isolated from the lysozyme-producing S. coelicolor clones obtained in Example 28 and Example 27, respectively, by the method described in D, A et al. 3 and 4). Plasmid pcell (Figure 3)
S, coelico was inserted into vector pGM4 which had been cut with BamHI and the overhanging ends had been filled in.
lor DSM 3030 total DNA, S.

coelicolor DSM 3030 Lysozyme (
Contains a 3.3 kb Small fragment encoding lys).

Vector 1) 0M4 contains the thiostrepton resistance gene (tsr) from S. azureus and S. fr.
neomycin resistance gene (a p b
I). Plasmid pce12 (Figure 4) is
S, coelicolor DSM 3 cloned into vector pEB 15 cut with Bgl[I
030 total DNA, S, coelicolor D
2, encoding SM 3030 lysozyme (lys);
Contains a 9kb 5au3A fragment. Vector pE
G15 is S.

The tyrosinase gene from Antibioticus (
tnel) and S. azureus, including the thiostrepton resistance gene (tsr).

Example 31 S, 1ivi of a plasmid encoding lysozyme
Transfer of plasmids pcell and pce12 into various S. dans and various S. coelicofor strains (Example 30
S, coelicolor 05M 3 obtained as in Example 23 and Example 24, respectively)
The 030 lysothyme deletion mutant was transformed (see Example 25). 50 mmol/liter HE
In a subsequent shaking culture test (see Example 23) in a 3M nutrient solution supplemented with PES, pH 7.0, and 30 Mg/ml thiostrepton, all of the transformants examined Type S.

The lysozyme productivity corresponding to the lysozyme production of coelicolor DSM 3030 was determined using the photometric test method (D
E 3440735).

C, J, Thompson et al. [J, Bacteri
The two plasmids were respectively transformed into S and
l 1vidansT K E34 (Hopwoo
d et al.: p. 266, supra). However, the isolated transformants were 50 mmo
l/liter HEPES (pH 7,0) and 30
Ca5o each containing Mg/ml thiostrepton.
Photometric test method (DE 34407) either in nutrient solutions (see Example 25) or in SM nutrient solutions.
35) showed no detectable lysozyme production.

On the other hand, plasmid pcel isolated as in Example 30
S by one of l or pce12, coelic
When transforming olor DSM 3030, use 50 mmol/liter of HEPES (pH 7
. The same increase in lysozyme production is achieved in SM medium in the absence of the antibiotic thiostrepton.

Example 32 Stability/Test Plasmid pce12 (see Example 30) containing S. coelicofor DSM 3030, 5
Shake at 30° C. for 10 days in an antibiotic-free SM nutrient solution containing 0 mmol/liter HEPES (pH 7,0). Each sample is appropriately diluted and plated onto five nutrient medium plates containing SM nutrient medium with or without 30 ttg/ml thiostrepton. After 7 days of incubation at 30° C., the same bacterial titer is found on the two types of plates, respectively. Transfer 500 colonies from each of the SM Nutrient Medium plates without thiostrepton to SM Nutrient Medium plates containing 3071 g/ml thiostrepton. 7 for 30
After incubation for days, it was possible to find the same number of colonies on both types of plates.

Example 33 Streptomyces before lysozyme gene from S. coelicolor DSM 3030
Cloning of the promoter region of the tyrosinase gene from C. antibiotics Plasmid pce12 (Fig. 4) isolated as in Example 30 was completely cleaved with restriction endonuclease A V a I, and the overhanging end was digested with E. coli DNA polymerase T (
frenow fragment) (Maniatis T, et al.:
Fill in the previous page, p. 3). Obtained 1,2kbDN
Example 21 of A fragment? Isolate using the method described above.

The vector plasmid pE B15 (see Example 26) is completely cut with the enzyme BgIII, the overhangs filled in, and dephosphorylated (see Example 2) to create the pc
A 1.2 kb sized isolated DNA fragment from e12 (see Example 3) is ligated. S with ligase mixture, coelicolor DSM 4
913 (see Example 25),
The resulting transformants were transferred to agar blocks containing 5M nutrient medium supplemented with 3071 g/ml thiostrepton (see Example 23) and incubated at 30°C in a humidified cabinet.
Incubate for days. The blocks are then transferred onto plates containing Micrococcus test agar (see Example 2) and incubated for 16 hours at 30°C in a humidified cabinet. Blasumi 1' DNA,
Restriction endonucleases were isolated from lysozyme-producing transformants, as observed by the formation of a cloudy melting zone on the test agar, as described in D.A., Hopwood et al., supra, p. 85. PvuU to completely disconnect. Plasmid pCe13 (Figure 4) containing the 2.1 kb and 2 kb PvuJI fragments is isolated.

The position direction of the lysozyme gene (tys) is pce13
Depending on the size of the pVuU fragment obtained, the fourth
Established as shown. Tyrosinase gene (met)
The promoter region of pce13 along with its promoter and signal sequence is placed in front of the lysozyme gene of pce13. S in plasmid pce13.

coelicolor DSM 3030 (C, J,
Thompson et al.: 1982, J. Bacte
riol, 151.668-677).

The transformant thus obtained is 50 mmol/
Liters of HEPES (r) B7. 30μ including O)
In a shaking culture test (see Example 23) with SM medium supplemented with and without thiostrepton at g/ml, in each case the starting strain S, coelicol
or DSM 3030 shows 3 to 5 times higher azozyme productivity.

It is a ward.

FIG. 4 is an explanatory diagram illustrating vector pEB15.

Claims (1)

[Claims] 1.a) The following sequence 5'-TTCGC(G/C)TACATCAAGGC(
G/C)AC(G/C)GAGGG(G/C)AC(G
/C) hybridizes with the oligonucleotide probe of AACTACAA-3', and/or b) after transformation, the lysozyme deletion mutant Streptomycescoelicolor DS
Lysozyme gene from Streptomyces complements M4913. 2. Streptomyces labedae or S.
treptomycescoelicolor (M■l
2. The lysozyme gene according to claim 1, which is derived from . 3.S. labedae DSM41517 or S. Claim 2 from coelicolor DSM3030
The lysozyme gene described in . 4. The lysozyme gene according to any one of claims 1 to 3, located on the 1.2 kb Aval fragment. 5. The lysozyme gene according to claim 4, which has the sequence shown in Table 1. 6. Lysozyme gene having the sequence shown in Table 2. 7. The lysozyme gene according to claim 6, wherein a heterozygous control or signal sequence is located upstream or downstream. 8. Streptomyces antibiotics
The lysozyme gene according to claim 7, wherein the promoter region of the tyrosinase gene from S. is located upstream. 9. A hybrid vector comprising the lysozyme gene according to any one of claims 1 to 8. 10. A microorganism comprising the hybrid vector according to claim 9. 11. Lysozyme expressed by the microorganism according to claim 10. 12. Lysozyme deletion mutant Streptomyc
escoelicolor DSM4913. 13. Oligonucleotide probe having the following sequence. 5'-TTCGC(G/C)TACATCAAGGC(
G/C)AC(G/C)GAGGG(G/C)AC(G
/C) AACTACAA-3'14, plasmid pEB
S.15 including coelicolor DSM4914.